This application relates to the detection of substances derived from biological fluids or tissue tagged with fluorochromes. It detects antigens, antibodies, hormones, enzymes, drugs and other substances.
Most infectious diseases of bacterial or viral nature produce antibodies in the blood serum of the subject. This provides a degree of immunity against future assaults by the identical infectious agent or antigen. One method for detecting the presence of a particular antigen is to add to it a specific antibody which binds to the antigen. If the antibody has been previously tagged with a radioactive element (RIA technique) or a fluorescent dye, which does not interfere with its immunological properties, the coupled complex can be detected by an appropriate detector and, in the case of the fluorescent additive, can be at best semiquantitatively measured, measuring being done in almost all cases in the prior art on a microscope slide for visual inspection.
As noted, testing which relies on fluorescence techniques, as heretofore known, involves qualitative assay, or at best, semiquantitative assay. For example, most fluorescence techniques occur on a microscope slide, and the detector is the eye of a laboratory technician who records the degree of fluorescence as 0, +1, +2, +3, or +4. In some instances, where blood titre or concentration of antibodies is desired, the technician prepares a number of slides; on each is a different concentration of the test material. Thus, the technician may estimate a +4 reaction in the microscope when the blood serum or the bacteria broth medium was diluted 1:4 in distilled water, or 1:16, or 1:128, etc. It would be of great advantage to medical and clinical authorities if a fluorometer could automatically and quantitatively read titre quickly and accurately, without the necessity of making serial dilutions.
Conventional fluorometers are designed for liquid systems and not capable of detecting fluorescently tagged substances derived from biological fluids or tissues on solid surfaces. One reason for this deficiency is the excessive loss (e.g., substantially over 99%) of emitted fluorescent light between the fluorescent substance and that portion of the detector which converts the light into an electrical signal.
There are many reasons why RIA is not completely satisfactory. For one thing, the presence of small quantities of antigen means few counts per second. Since the "noise" of the system is the square root of the signal count, large errors in accuracy are made at these low signal levels. Furthermore, radioisotopes have a limited shelf life due to half life decay, and require special licensing, handling and disposal.